We sought to identify, purify and partially characterize a protein inhibitor of Na + /K + -ATPase in cytosol of pulmonary artery smooth muscle.(i) By spectrophotometric assay, we identified an inhibitor of Na + /K + -ATPase in cytosolic fraction of pulmonary artery smooth muscle; (ii) the inhibitor was purified by a combination of ammonium sulfate precipitation, diethylaminoethyl (DEAE) cellulose chromatography, hydroxyapatite chromatography and gel filtration chromatography; (iii) additionally, we have also purified Na + /K + -ATPase α 2 β 1 and α 1 β 1 isozymes for determining some characteristics of the inhibitor.We identified a novel endogenous protein inhibitor of Na + /K + -ATPase having an apparent mol mass of ∼70kDa in the cytosolic fraction of the smooth muscle. The IC 50 value of the inhibitor towards the enzyme was determined to be in the nanomolar range. Important characteristics of the inhibitor are as follows: (i) it showed different affinities toward the α 2 β 1 and α 1 β 1 isozymes of the Na + /K + -ATPase; (ii) it interacted reversibly to the E 1 site of the enzyme; (iii) the inhibitor blocked the phosphorylated intermediate formation; and (iv) it competitively inhibited the enzyme with respect to ATP. CD studies indicated that the inhibitor causes an alteration of the conformation of the enzyme. The inhibition study also suggested that the DHPC solubilized Na + /K + -ATPase exists as (αβ) 2 diprotomer.The inhibitor binds to the Na + /K + -ATPase at a site different from the ouabain binding site. The novelty of the inhibitor is that it acts in an isoform specific manner on the enzyme, where α 2 is more sensitive than α 1 .